Opioids are used extensively to relieve pain but also produce detrimental effects, including opioid-induced hyperalgesia (OIH) and analgesic tolerance. OIH and tolerance reduce opioid efficacy and drive dose escalation, which worsens other deleterious effects such as respiratory depression, and transition to addiction. These effects dramatically impact the quality of life of pain patients. Until now it has not been possible to dissociate the analgesic from the OIH and tolerance effects, because the receptors and cells on which opioids act to cause OIH and tolerance have not been identified. In contrast to the current model of opioids acting on CNS microglia to initiate antinociceptive tolerance and OIH, we found in RNA-sequencing experiments that the mu opioid receptor (MOR) is not expressed by microglia, and that OIH is lost, but microglia activation intact, in MOR global knockout mice. Furthermore, we generated new mutant mice that lack MOR only in dorsal root ganglion (DRG) nociceptors, but have intact MOR function in the CNS, and found that these mice show intact morphine antinociception, but no OIH or tolerance. These preliminary studies open the possibility of dissociating opioid analgesia from side effects, and lead to the following hypothesis: MORs in CNS underlie opioid analgesia, while MOR signaling and maladaptive neuroplasticity in DRG is responsible for OIH and tolerance. In Aim 1, we will use mouse genetics and viruses to delete MOR only in DRG of mice with chronic pain and treated with opioids, and submit these mice to behavioral assays to measure opioid analgesia, analgesic tolerance, and OIH, including with measures of pain affect and spontaneous pain. We will also block peripheral MORs with FDA- approved peripherally restricted antagonist methylnaltrexone bromide. We predict that deleting or blocking MOR in DRG will reduce morphine tolerance and OIH without impacting analgesia. In Aim 2, we will resolve the maladaptive synaptic mechanisms that underlie OIH and tolerance. Opioids induce pronociceptive long-term potentiation (LTP) at the synapse between DRG and spinal neurons. We will combine optogenetics and electrophysiological analysis to determine whether activation of presynaptic MORs in DRG initiates maladaptive LTP. In Aim 3, we will use RNA-sequencing on individual DRG nociceptors and PZM21, a novel Gi-biased MOR agonist, to determine the MOR effectors present in identified nociceptors, the relevance to tolerance/OIH of Gi versus beta-arrestin signaling in DRG neurons, and how opioids alter the expression of the genes controlling DRG neuron excitability and neurotransmission. This research will transform our understanding of the mechanisms of action of opioids by identifying MOR in DRG as the target of opioids for OIH and tolerance. This research also has the potential to transform clinical practice, as MNB, or other drugs acting on targets identified by RNA-sequencing, might be used to treat pain at lower opioid dosage. In the future, the approach established here may help uncover the mechanisms underlying other opioid side effects, such as respiratory depression and addiction, to identify innovative strategies to limit transition to addiction and death by overdose from opioid abuse.